Human APOBEC3H (A3H) has one cytidine deaminase domain (CDD) and inhibits the replication of retrotransposons and human immunodeficiency virus type 1 (HIV-1) in a Vif-resistant manner. Human A3H has five single amino acid polymorphisms (N15⌬, R18L, G105R, K121D, and E178D), and four haplotypes (I to IV) have previously been identified in various human populations. Haplotype II was primarily found in African-derived populations, and it was the only one that could be stably expressed. Here, we identified three new haplotypes from six human population samples, which we have named V, VI, and VII. Haplotypes V and VII are stably expressed and inhibit HIV-1 replication. Notably, haplotype V was identified in samples from all African-, Asian-, and Caucasian-derived populations studied. Using haplotype VII, we investigated the A3H anti-HIV-1 mechanism. We found that A3H virion packaging is independent of its CDD but dependent on a 112 YYXW 115 motif. This motif binds HIV-1 nucleocapsid in an RNA-dependent manner, and a single Y112A mutation completely disrupts A3H virion incorporation. We further studied the mechanism of A3H resistance to Vif. Although the previously identified APOBEC3G Vif-responsive motif 128 DPDY 131 is not conserved in A3H, placement of this motif into A3H does not make it become less resistant to HIV-1 Vif. We conclude that stably expressed A3H haplotypes may be more broadly distributed in humans than previously realized, and A3H protein is resistant to Vif. These results have important implications for the role of A3H in retrotransposon and HIV-1 inhibition.

During studies of APOBEC3 (A3) anti-human immunodeficiency virus type 1 (anti-HIV-1) mechanisms, we identified a single cysteine at position 320 (C320) that disrupts A3DE activity. This residue is located in the recently identified DNA binding domain in A3G. Replacing C320 with a corresponding tyrosine from A3F (Y307) increased A3DE antiviral activity more than 20-fold. Conversely, replacing A3F Y307 with a cysteine or inserting a similar cysteine into A3B or A3G disrupted the anti-HIV activity of A3. Further investigation uncovered that C320 significantly reduces A3DE catalytic activity.The A3DE gene was originally proposed as two genes, i.e., separated A3D and A3E genes (8). Later, we and others found that they are expressed as a single gene (4, 13), which was renamed A3DE (4). Despite sharing high homology with other A3 proteins that have two cytidine deaminase (CDA) motifs, A3DE exhibits relatively low levels of anti-human immunodeficiency virus type 1 (anti-HIV-1) activity, and Vif still neutralizes this activity (4). To map the domain responsible for its low activity, we used overlap extension PCR to create three , and A3F are shown in orange, blue, and green, respectively. Three variable regions I, II, and III are indicated. (C) Alignment of A3DE and A3F CTD amino acid sequences. Conserved residues are shown in red, nonconserved residues are shown in black, and residues in the CDA motif are shown in blue. Three C-terminal variable regions (I, II, III) are boxed, and both A3DE C320 and A3F Y307 residues are indicated. Secondary structure elements used in making the A3DE model in Fig. 3A are shown on top of the sequence. (B and D) Anti-HIV-1 activity of A3 chimeras and A3DE mutants. ⌬Vif HIV-1 luciferase reporter viruses were produced in 293T cells in the presence of each of these proteins, and their infectivity was determined after infection of GHOST cells. The value of infectivity was calculated from quantitation of luciferase enzyme produced in the GHOST cells, after normalization by levels of viral input (p24 Gag ). The infectivity of virions produced in the presence of A1 was considered 100%, and others were calculated as relative values according to this standard. The expression of indicated proteins in 293T cells was also determined by Western blotting (lower panels). The standard errors of the means (SEM) were calculated from three independent experiments. 5691on May 12, 2018 by guest